The present invention relates to a nuclear magnetic resonance measurement apparatus and a measuring method using the same and more particularly, to a technique effectively applicable to the nuclear magnetic resonance measurement of a high molecular compound such as protein or macromolecule.
Such a molecule as protein functioning in a living body has a larger molecular weight in comparison with a low molecular compound of molecular weight 1000 or less used for medicine and the like and has characteristics of a high molecular compound. The high molecular compound typified by the protein functions molecularly in a solution and the molecular function is either hindered or promoted when the high molecular compound combines with a specified low molecular compound. The combination or interaction of the specified low molecular compound with the high molecular compound can be detected through many methods.
Especially, in the nuclear magnetic resonance measurement (hereinafter referred to as NMR measurement) capable of directly observing information about a molecular structure of the high molecular compound or the low molecular compound, not only the dissociation constant and reaction rate the high molecular compound and low molecular compound have can be evaluated from how the measuring spectrum changes with the compound concentration but also the interaction can be analyzed on the basis of the structure of the compound. JP-A-2003-510608 (Patent Document 1) describes a method of measuring the interaction between protein and low molecular compound through the NMR measurement.
Of the high molecular compounds used for the NMR measurement, the protein can be formed through a method of extracting it from a living body existing in the natural world, a method of extracting it from a mass developing system that takes advantage of, for example, colitis germs built in with a gene associated with formation of the target protein or a method of utilizing a non-cell developing system that develops a large amount of proteins without using living cells. Further, in some case, principal elements constituting the protein such as hydrogen, carbon and nitrogen are marked (hereinafter termed labeled) with isotope radioactive elements. Available in the labeling are a method of performing labeling by using the three elements of hydrogen, carbon and nitrogen constituting the protein in combination, a method of total marking for labeling all elements and a method of selective marking for labeling only atoms belonging to a specified amino acid residue. In any methods, the cost of labeling process is high.
Typically, the nuclear magnetic resonance measurement apparatus (hereinafter referred to as NMR measurement apparatus) comprises a magnet for generating a statistic magnetic field (Bo) and a nuclear magnetic resonance probe placed in a space inside the magnet. The nuclear magnetic resonance probe includes one or more coils adapted to apply an RF magnetic field (B1) to a target sample and detect a reaction (response) the sample makes to the applied RF magnetic field.
The conventional nuclear magnetic resonance probe is sorted into a stationary sample probe and a flow-through probe. In the stationary sample probe, a method is adopted in which a sample is fed into a glass tube or an ampule (hereinafter referred to as a sample tube), the sample tube is set at a predetermined location in the NMR measurement apparatus and thereafter measurement is started.
In titration measurement using the conventional stationary sample probe, NMR measurement is carried out while titrating a low molecular compound with the help of a sample tube having an opening, so that how an NMR spectrum changes with an increase in concentration can be observed. But a sample solution is a mixture solution containing a high molecular compound, a low molecular compound representing a medicine effect evaluation target and other reagents and therefore, once the sample solution has reached a condition of containing the low molecular compound at a certain concentration, the NMR measurement is difficult to proceed by using that low molecular compound under a condition of lower concentration.
Further, because of the volume of the sample tube being small, as the titration of the low molecular compound proceeds and the volume of the total sample solution increases relatively, the concentration of the high molecular compound sample changes and besides the liquid level of the sample solution changes. In order to suppress these changes, the volume to be titrated needs to be reduced as far as possible in relation to the volume of the sample solution. But for reduction of the titration volume, the concentration of the low molecular compound is required to be increased. The concentration of a titration solution has, however, its upper limit that is determined by the solubility of the low molecular compound and in general, the solubility of substance differs remarkably with the kind of a solvent and the temperature.
As described above, in the adjustment of low molecular compound concentration through titration, the sample volume varies. The variation changes in its magnitude with the combination of low molecular compound and high molecular compound, the solvent and the temperature. The concentration change is unidirectional.
On the other hand, the conventional flow-through probe includes a sample inlet, a sample outlet and an internal tube extending between the inlet and the outlet. The internal tube contains a cell for holding a sample. A sample flows from the sample inlet to enter the cell through the internal tube. After measurement, the sample flows through the tube so as to be discarded externally of the probe.
The conventional flow-through probe has been used in combination with a robot-type sample conveyance system. In the system, samples adjusted for a plurality of measurement conditions need to be prepared in advance in a plurality of reservoirs. The sample is conveyed, via a unit capable of bringing out the sample, from a reservoir to a flow-through probe set in advance. JP-A-2004-534958 (Patent Document 2) describes the sample conveyance system.
In the combination of the conventional flow-through probe with the sample conveyance system, samples adjusted for the plurality of concentration conditions are required to be prepared in advance. Accordingly, high molecular compound solutions of predetermined concentrations are necessary correspondingly to the number of measurement conditions and high costs are required of the samples.
In the case of high molecular compound and low molecular compound for which the measurement condition range is unknown, the whole of measurement of function evaluation must be repeated until a preferable range of measurement conditions and the degree of changes in conditions are settled, raising a troublesome problem. JP-A-2007-315826 (Patent Document 3) describes a system capable of performing repetition of NMR measurement by utilizing a simplified circulation channel.
[Patent Document 1] JP-A-2003-510608
[Patent Document 2] JP-A-2004-534958
[Patent Document 3] JP-A-2007-315826